Plasmodium vivax (Pv), the second most important human malaria parasite, causes more than 80 million cases annually including severe, fatal disease. Prevention and control are challenged by emerging drug resistance and relapses from dormant liver stage parasites called hypnozoites. The only therapy against relapse, primaquine, causes life threatening acute hemolytic anemia in patients with G6PD deficiency, the most prevalent human genetic disorder, affecting 8% of people in malaria-endemic nations. This barrier to treatment results in repeated Pv attacks, aggravating the problem of control. The demonstration of high level (100%), long-lasting (at least 12 months) protective efficacy of Sanaria's sporozoite (SPZ)-based vaccines against Pf malaria is a significant milestone, and indicates that such vaccines will constitute a viable approach to containing and eliminating Pf. We believe that the same vaccine approach could work for Pv. In the development of the Pf vaccines, controlled human malaria infection (CHMI) has been an engine of progress, accelerating the testing of vaccine candidates. Pf CHMI has recently been revolutionized by the development of Sanaria's PfSPZ Challenge, aseptic, purified, cryopreserved, fully infectious PfSPZ derived from in vitro cultures of Pf gametocytes, enabling the successful infection of volunteers by intradermal, intramuscular, and intravenous injection in 3 countries in Africa and 2 countries in Europe that had never conducted CHMI before. In contrast, development of Pv SPZ-based products has suffered from lack of available technology to culture Pv parasites in vitro, such that generating infected mosquitoes for CHMI required membrane feeding on fresh, Pv-infected blood from Pv patients. We have now overcome this major limitation by using Pv gametocyte- infected Saimiri boliviensis non-human primates (NHPs) to produce PvSPZ. In fact we are the only laboratory with an inventory of vialed PvSPZ made from NHP-infected blood, having produced as much as 80 million PvSPZ vialed in 1 day from 2,000 mosquitoes. These cryopreserved PvSPZ are 1) infectious to hepatocyte cell lines in vitro in traditional monolayer formats over 3-6 days and in micro-patterned co-cultured primary human hepatocytes over 12-21 days, and 2) infectious to NHPs in vivo. We now propose to produce aseptic, purified, cryopreserved, infectious PvSPZ (PvSPZ Challenge) by using a specific germ free colony of the permissive S. boliviensis as the source for Pv-infected blood. This novel pipeline will generate cGMP-compliant, controlled batches of PvSPZ including a wide variety of primary and clonal Pv lines isolated from humans. This innovation by Sanaria will offer a consistent, quality-controlled stock of cryopreserved PvSPZ to promote well-controlled, reproducible in vitro and in vivo studies in Pv including CHMI. This enabling technology will support the development and testing of anti-Pv drugs and vaccines in CHMIs world-wide, just as PfSPZ Challenge has done for Pf CHMIs. It will also form the basis of a powerful vaccine approach to preventing Pv malaria when administered with anti-malarial chemoprophylaxis, the PvSPZ chemoprophylaxis vaccine (PvSPZ-CVac).